Several observations indicate that CD8+ T cells are important in controlling virus replication during chronic HIV and SIV infection. Paramount among these observations is that experimental in vivo depletion of CD8+ lymphocytes in SIV-infected rhesus macaques (RM) is followed by a dramatic increase in virus replication. However, the mechanisms by which CD8+ T cells mediate this antiviral effect are still poorly understood, as emphasized by the negative result of the Merck Adenovirus-based, cytotoxic T lymphocyte (CTL)-inducing candidate AIDS vaccine in a large phase IIb clinical trial. In a recent study (Klatt et al., PLoS Pathogens, in press), we sought to assess the mechanisms underlying the antiviral effect of CD8+ lymphocytes during chronic SIVmac239 infection of RMs by treating two groups of animals (i.e., CD8+ lymphocyte-depleted or controls) with antiretroviral therapy (ART). Using a well-accepted mathematical model to calculate the in vivo lifespan of productively infected cells, we found that, in both early and late SIV infection, depletion of CD8+ lymphocytes did not change the in vivo lifespan of infected cells. This result indicates that SIV suppression mediated by CD8+ lymphocytes goes above and beyond the direct killing of cells producing virus, and suggests that more studies are needed to fully understand the antiviral role of CD8+ T cells during SIV infection. Here we propose to conduct a series of experiments of in vivo CD8+ lymphocyte depletion aimed at better elucidating the antiviral effects of these cells during chronic SIV mac239 infection of RMs. In the first Aim, we will determine how CD8+ lymphocytes impact the average lifespan of in vivo infected cells in Mamu-B*08/17+ RMs that show better control of virus replication upon SIV mac239 infection. In the second Aim, we will determine how CD8+ lymphocyte depletion induces changes in the fraction, type, and location of SIV infected cells as well as the burst size of virus production. In the third Aim, we will assess how CD8+ lymphocyte depletion affects the production of soluble antiviral factors (i.e., cytokines and chemokine) and alters the intrinsic susceptibility of CD4+ T cells to SIV infection by changing their level of activation, proliferation, CCR5 expression, and intracellular levels of host restriction factors. We believe that these experiments will provide important insights into CD8+ lymphocyte inhibition of virus replication during SIV infection. Ultimately, we hope that this knowledge will favor the rational design of an effective CTL-based AIDS vaccine. Our current inability to design immunogens that induce broadly reactive neutralizing antibodies against the HIV-1 Envelope protein has shifted the focus of the AIDS vaccine development effort to the design of immunogens that induce high levels of HIV-specific CD8+ T cells. The experiments included in this proposal are aimed at elucidating the mechanisms by which CD8+ lymphocytes suppress virus replication in SIV-infected RMs. The results generated here will provide correlates of CD8+ lymphocyte-mediated protection to be used to rank and prioritize the development of candidate AIDS vaccines for use in humans.